Lignin, a complex phenylpropanoid polymer, is the second most abundant natural product after cellulose. In trees, lignin may contribute up to about 20-30% of the dry weight. Lignin is primarily deposited in the cell walls of supporting and conductive tissues, such as fibers and tracheary elements. The mechanical rigidity of lignin strengthens these tissues so that the tracheary elements can endure the negative pressure generated from transpiration without collapse of the tissue. In addition to providing mechanical strength, lignin has significant protective functions. Both the physical toughness and chemical durability of lignin may deter feeding by herbivores. Lignification is a frequent response to infection or wounding, which may provide a physical barrier to block the penetration of pathogens.
However, lignin is undesirable in several aspects. Lignin decreases the digestibility of animal forage crops and must be removed during pulping and paper making, which requires the use of chemicals hazardous to the environment. Lignin also appears to have a negative impact on the utilization of plant and tree biomass. During paper pulping, lignin must be eliminated from wood by chemical treatments that are expensive and polluting. The quantity of lignin in forage crops is also negatively correlated to digestibility and appetence. It has been suggested that even small improvements in the digestibility of forage crops or in the reduction of chemicals used for kraft pulping would be valuable because of the large scale of the industries (Sederoff et al., Genetic Engineering of Plant Secondary Metabolism, New York Plenum Press (1994)).
Lignin is derived from the dehydrogenative polymerization of monolignols; notably, p-coumaryl alcohol, coniferyl alcohol and synapyl alcohol. Lignin is heterogeneous in that it is composed of different proportions of these three monolignols in different plant species tissues, cell types and even in the same cell at different developmental stages. For example, gymnosperm lignin is primarily composed of guaiacyl (coniferyl-derived) units, whereas angiosperm dicot lignin is primarily composed of guaiacyl and syringyl (synapyl-derived) units. Grass lignin is typically a mixture of guaiacyl, syringyl and p-hydroxylphenyl (coumaryl-derived) units. At tissue and cell type levels, energy dispersive X-ray analysis has shown that the ratios of guaiacyl to syringyl lignin are 12:88 and 88:12 in fibers and vessels, respectively, of birch wood (Saka et al., Holzforschung, 42:149-153 (1988)). Lignin heterogeneity is likely to be controlled by the differential expression of lignin enzymes in different lignified tissues.
Monolignols are synthesized through a phenylpropanoid biosynthetic pathway. The chemical structures of the various monomeric lignin precursors, for example, p-coumaric acid, ferulic acid and sinapic acid, differ typically in the number and position of methoxy groups on the aromatic ring. Thus, the methylation of 3- and/or 5-hydroxyl groups of hydroxycinnamic acids in the biosynthetic pathway is an important step influencing lignin composition. It is well accepted that the methylation pathway is mediated by the enzyme caffeic acid O-methyltransferase (CAOMT). The CAOMT-mediated methylation pathway uses free acids as intermediates.
In 1968, Neish proposed an alternative methoxylation pathway in which the carboxyl group is first activated either on cinnamic acid or on p-coumaric acid, and subsequent hydroxylation and methylation are carried out on these ester forms instead of on the free acids Neish, Constitution and Biosynthesis of Lignin, Springer Verlag (1968)). Recently, it was suggested that a methylation pathway mediated by caffeoyl-CoA O-methyltransferase (CCoAOMT) is involved in lignin biosynthesis (Ye et al., Plant Cell, 6:1427-1439 (1994)). The association of CCoAOMT with lignification was demonstrated in a number of dicot plants (Ye et al., Plant Physiol., 108:459-467 (1995); Ye, Plant Physiol., 115:1341-1350 (1997)). However, no direct evidence of a role for CCoAOMT in lignification has been identified, and the CCoAOMT-mediated methylation pathway has not been widely accepted due to a lack of genetic evidence supporting such a pathway. A significant reason for the general lack of acceptance of CCoAOMT participation in lignification has been the finding that reduction in CAOMT enzyme activity alone in transgenic plants effectively blocks syringyl lignin production (Atanassova et al., Plant J., 8:465-477 (1995); Van Doorsselaere et al., Plant Journal, 8:855-864 (1995); Tsai et al., Plant Physiol., 117:101-112 (1998); Dwivedi et al., Plant Mol. Biol., 26:61-71 (1994)). Thus, doubts have been cast on the role of CCoAOMT in lignin production.
A reduction of lignin content and/or alteration of lignin composition is desirable in that it would reduce the pollution from pulping and improve the digestibility of animal forage. It is therefore of interest to develop methods that allow for the modification of lignin content and/or composition of a particular plant, tree or grass cell and /or tissue for producing plants having reduced lignin content.
The present invention provides a plant characterized by reduced lignin content and/or altered lignin composition compared to a wild-type plant. Plants of the invention include, for example, gymnosperms, angiosperms or forage crops such as alfalfa, tall fescue and clover. Preferably, the plant is a genetically engineered plant.
Preferably, the plant of the invention exhibits reduced activity of at least one biosynthetic enzyme involved in lignin biosynthesis, compared to the activity of the biosynthetic enzyme in the wild-type plant. In a particularly preferred embodiment, the plant exhibits reduced activity of a caffeoyl-CoA O-methyltransferase enzyme. Optionally, the plant additionally exhibits reduced activity of a caffeic acid O-methyltransferase enzyme compared to the caffeic acid O-methyltransferase enzyme activity of a wild-type plant.
In a preferred embodiment, a plant is genetically engineered in a way that makes use of antisense technology to effect a reduction in the activity of a biosynthetic enzyme. Accordingly, a preferred plant of the invention contains at least one exogenous nucleic acid comprising a nucleotide sequence that is xe2x80x9cantisense toxe2x80x9d at least a portion of a caffeoyl-CoA O-methyltransferase gene such that when the exogenous nucleic acid is present, the activity of an endogenous caffeoyl-CoA O-methyltransferase enzyme is inhibited. Another preferred plant contains at least two exogenous nucleic acids, a first comprising a nucleotide sequence that is antisense to at least a portion of a caffeoyl-CoA O-methyltransferase gene and a second comprising a nucleotide sequence that is antisense to at least a portion of a caffeic acid O-methyltransferase gene such that when the first and second exogenous nucleic acids are present, the activities of both an endogenous caffeoyl-CoA O-methyltransferase enzyme and an endogenous caffeic acid O-methyltransferase enzyme are inhibited. The exogenous nucleic acids can be present in the plant as part of the same nucleic acid molecule (for example, as when they are present on the same vector); or they can exist as separate molecules (for example, as when they are present on different vectors).
Methods for making the plants of the invention are also provided. Plants are genetically or biochemically engineered to reduce the activity of one or more enzymes involved in the phenylpropanoid biosynthesis pathway of the plant. In one embodiment, the invention provides a method for making a genetically engineered plant that includes transfecting a plant cell with at least one exogenous nucleic acid associated with reduced activity in the plant of at least one biosynthetic enzyme involved in lignin biosynthesis, followed by growing the transfected plant cell into the genetically engineered plant having reduced lignin content compared to the lignin content of a comparable wild-type plant. Preferably, the biosynthetic enzyme is CCoAOMT. In a preferred embodiment, the exogenous nucleic acid includes a nucleotide sequence that is xe2x80x9cantisensexe2x80x9d to at least a portion of a CCoAOMT gene. Optionally, the plant cell can be additionally transfected with a second exogenous nucleic acid, for example one that is xe2x80x9cantisensexe2x80x9d to at least a portion of a CAOMT gene. The genetically engineered plant cell is then grown into a plant characterized by reduced lignin content and/or altered lignin composition compared to a wild-type plant.
When antisense technology is used to practice the invention, exogenous nucleic acids are advantageously supplied to the plant cell in the form of one or more expression vectors. Plant cells containing two or more different antisense nucleic acids can be made by transfecting the plant cell with a single vector that encodes all the desired antisense molecules, or by a plurality of vectors, each encoding one or more antisense nucleic acids. Multiple copies of a single antisense nucleic acid can, but need not be included on a single vector. Preferably, each copy of an antisense nucleic acid is operably linked to its own promoter and terminator.
The invention further provides a method for making a genetically engineered plant having altered lignin composition compared to the lignin composition of a comparable wild-type plant. A plant cell is transfected with at least one exogenous nucleic acid associated with altered (i.e., either increased or decreased) activity in the plant of at least one biosynthetic enzyme involved in lignin biosynthesis. The transfected plant cell is then grown into the genetically engineered plant having altered lignin composition.